The present invention provides known and novel compounds, their use in the inhibition of an enzyme whose preferred mode of action is to catalyse the hydrolysis of an ester functionality (in vivo, as the enzyme naturally occurs) their use in medicine, and particularly in the prevention and/or treatment of obesity or an obesity-related disorder. Also provided are methods for the prevention and/or treatment of obesity or an obesity-related disorder and for promoting/aiding non-medical weight loss and the use of the compounds in the manufacture of a medicament for the aforementioned indications. In respect of novel compounds the invention also provides processes for their manufacture, compositions containing them and methods for manufacturing such compositions.
In the last 20 years, there has been an increasing trend in obesity in the populations of the developed world. The increased incidence of obesity is due in part to the ready availability of food in numerous retail outlets and westernised diets that have high saturated fat and lower fibre contents such that the food is energy dense. The lifestyle of the populations of the developed world has also become more sedentary with the increased mechanisation of society and the steady reduction of manual labour intensive industries. There now exists an energy imbalance between the energy intake from calorie dense foods and the reduced energy expenditure required for a sedentary lifestyle. Some of the excess energy intake is stored as fat in the adipose tissue, the accumulation of which over a period of time results in obesity and can be a significant contributory factor to other disease and disorders.
Obesity is now recognised by the medical profession as a metabolic disease. In the USA, it is estimated that 25% of the adult population is considered clinically obese (Body Mass Index greater than 30). Obesity can be a debilitating condition which reduces the quality of life and increases the risk of related disorders such as diabetes, cardiovascular disease and hypertension. It has been estimated that $45 billion of US healthcare costs, or 8% per annum of total healthcare spend, is as a direct result of obesity. The traditional approaches to long term weight management such as diet and exercise have proved ineffective alone to control the spread of obesity. Today, more than ever, there is considerable interest in developing safe, effective drugs for the treatment of obesity.
Pharmacological approaches to the treatment of obesity have focused on either developing drugs that increase energy expenditure or drugs that reduce energy intake. One approach to the reduction of energy intake is to reduce the body""s ability to digest and absorb food, in particular fat. The key enzymes involved in the digestion of fat are hydrolytic enzymes. The most significant of the fat degrading enzymes are lipases, primarily, but not exclusively pancreatic lipase that is secreted by the pancreas into the gut lumen. The lipase inhibitor lipstatin has formed the basis of the anti-obesity drug, orlistat. Orlistat is the subject of published European Patent Application No. EP129748, which relates to compounds of formula: 
where A is xe2x80x94(CH2)5xe2x80x94 or; 
and their use in inhibiting pancreatic lipase and treating hyperlipaemia and obesity. Orlistat has as its major active moiety a beta-lactone group that reacts to form an ester with the side chain hydroxyl group of serine 152 within the active site of pancreatic lipase.
Even if orlistat provides an effective method for treating obesity, there remains a need to provide alternative drugs and methods for use in the control and treatment of obesity and obesity-related disorders and in promoting or aiding non-medical weight loss. Inhibitors of enzymes involved in the degradation of fat are provided here and shown to be effective in the prevention and/or treatment of obesity, obesity-related disease and/or in promoting cosmetic weight loss.
U.S. Pat. No. 4,665,070 (Syntex) describes a broad class of 2-oxy-4H-3,1-benzoxazin-4-ones of the formula: 
where a is an integer from 0-4; each Rxe2x80x2 may be selected from a wide range of substituents; A is a bond or a C1-8 alkylene group; and R is H (except when A is a bond) phenyl, imidazolyl or C3-6 cycloalkyl each of which rings may be optionally substituted. The Rxe2x80x2 groups are said to be preferably in the 5- and/or 7-positions of the ring. A preferred value of the group A is lower alkylene having 1-4 carbon atoms. In the most preferred compounds A is ethylene. The compounds are said to be useful as serine protease inhibitors and to treat physiologic conditions and disease states known to involve enzymes, or as contraceptives. The specification describes various conditions and diseases involving enzymatic pathways, including inflammation, arthritis, tumor cell metastasis, pulmonary emphysema, mucocutaneous lymph node syndrome, adult respiratory distress syndrome and pancreatitis. It is also suggested that the compounds may have antiparasitic, anticoagulant and/or antiviral activity. Similar compounds are also described in U.S. Pat. No. 4,745,116.
International Patent Application No. WO89/07639 (BP Chemicals Ltd) describes detergent compositions in aqueous solution which comprise a surfactant, a precursor compound capable of giving rise to a peroxygen compound in the presence of water, a suds suppressing agent, a detergent builder and a bleach activator which may have the formula: 
where R is inter alia an alkoxy group and R1, R2, R3 and R4 (which may be the same or different) are selected from H, halogen, alkyl, alkenyl, aryl, hydroxyl, alkoxyl, amino, alkylamino, xe2x80x94COOR5 and carbonyl functions. The number of carbon atoms in the alkyl groups and moieties is not defined, but the specific examples are of lower alkyl and alkoxy groups e.g. R may be ethoxy.
East German Patent No. DD 246996A1 describes a process for preparing 2-alkoxy- and 2-aryloxy-3,1-benzoxazin-4-ones of the formula: 
where Rxe2x80x2n represents one or more H atoms and/or other substituents such as alkyl, alkoxy, aralkyl, aryl, thiocyanato, mercapto, alkylthio, halogen or nitro, and R2 represents an alkyl, aralkyl or aryl residue. The compounds are said to be useful as herbicides and fungicides and to have activity as inhibitors of chymotrypsin. Specific examples of R2 are ethyl, benzyl and phenyl.
We have now found that a particular class of benzoxazinone compounds has activity as lipase inhibitors.
Accordingly, in a first aspect, the present invention provides the use of a compound comprising formula (I): 
or a pharmaceutically acceptable salt, ester, amide or prodrug therof; in the manufacture of a medicament for the treatment of conditions which require the inhibition of an enzyme whose preferred mode of action is to catalyse the hydrolysis of an ester functionality, wherein in formula (I):
A is a 6-membered aromatic or heteroaromatic ring; and
R1 is a branched or unbranched alkyl (optionally interrupted by one or more oxygen atoms), alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, arylalkyl, reduced arylalkyl, arylalkenyl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, reduced aryl, reduced heteroaryl, reduced heteroarylalkyl or a substituted derivative of any of the foregoing groups, wherein the substituents are one or more independently of halogen, alkyl, halosubstituted alkyl, aryl, arylalkyl, heteroaryl, reduced heteroaryl, reduced heteroarylalkyl, arylalkoxy, cyano, nitro, xe2x80x94C(O)R4, xe2x80x94CO2R5, xe2x80x94SOR4, xe2x80x94SO2R4, xe2x80x94NR6R7, xe2x80x94OR6, xe2x80x94SR6, xe2x80x94C(O)CX1X2NR6R7, xe2x80x94C(O)N(OH)R6, xe2x80x94C(O)NR5R4, xe2x80x94NR6C(O)R4, xe2x80x94CR6(NH2)CO2R6, xe2x80x94NHCX1X2CO2R6, xe2x80x94N(OH)C(O)NR6R7, xe2x80x94N(OH)C(O)R4, xe2x80x94NHC(O)NR6R7, xe2x80x94C(O)NHNR6R7, xe2x80x94C(O)N(OR5)R6, or a lipid or steroid (natural or synthetic) with the proviso that any hetero atom substituent in R1 must be separated from the exocyclic oxygen atom by at least two carbon atoms (preferably saturated);
and where:xe2x80x94
R4 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, reduced heteroaryl, reduced heteroarylalkyl, xe2x80x94OR6, xe2x80x94NHCX1X2CO2R6 or xe2x80x94NR6R7;
R 5 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, arylalkyl heteroaryl, heteroarylalkyl, reduced heteroaryl or reduced heteroarylalkyl; and
R6 and R7 are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, arylalkyl, heteroaryl, reduced heteroaryl, reduced heteroarylalkyl or xe2x80x94(CH2)n(OR5)m wherein n is 1 to 12, preferably 2 to 10, wherein m is 1-3 and R5 is most preferably C2-C10 alkyl; and
X1 and X2 are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, reduced heteroaryl or reduced heteroarylalkyl.
In compounds of formula (I) any alkyl, alkenyl and alkynyl groups and moieties may be straight chain (unbranched) or branched chain. Straight chain alkyl, alkenyl and alkynyl groups or moieties may contain from 1 to 30 carbon atoms, eg. 1 to 25 carbon atoms, preferably 1 to 20 carbon atoms. Branched chain alkyl, alkenyl and alkynyl groups or moieties may contain from 1 to 50 carbon atoms, preferably 1 to 30 carbon atoms.
Preferred values for R1, R4, R5, R6, R7, X1 and X2 are as defined below for formulae (II) and (IIa). In particular, preferred values for R4, R5 and R6 are as defined for R13 hereinbelow and preferred values for R7 are as defined for R14 hereinbelow.
In this text, xe2x80x98reducedxe2x80x99, in the context of xe2x80x98reduced heteroarylxe2x80x99 and the like means fully or partially saturated.
Aryl groups include for example optionally substituted unsaturated monocyclic or bicyclic rings of up to 12 carbon atoms, such as phenyl and naphthyl, and partially saturated bicyclic rings such as tetrahydro-naphthyl. Examples of substituents which may be present on an aryl group include one or more of halogen, amino, nitro, alkyl, haloalkyl, alkoxy, phenoxy and phenoxy substituted by one or more of halo, alkyl or alkoxy.
A heteroaryl group or moiety may be for example an optionally substituted 5- or 6-membered heterocyclic aromatic ring which may contain from 1 to 4 heteroatoms selected from O, N and S. The heterocyclic ring may optionally be fused to a phenyl ring. Examples of heteroaryl groups thus include furyl, thienyl, pyrrolyl, oxazolyl, oxazinyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyrimidinyl, pyrazolyl, indolyl, indazolyl, benzofuranyl, benzothienyl, benzimidazolyl, benzoxazolyl, benzoxazinyl, quinoxalinyl, quinolinyl, quinazolinyl, cinnolinyl, benzothiazolyl, pyridopyrrolyl. Suitable substituents include one or more of halogen, oxo, amino, nitro, alkyl, haloalkyl, alkoxy, phenoxy and phenoxy substituted by one or more of halo, alkyl, haloalkyl or alkoxy.
A reduced heteroaryl group or moiety may be for example a fully or partially saturated derivative of the aforementioned heteroaryl groups. Examples of reduced heteroaryl groups thus include pyrrolidinyl, tetrahydrofuryl, tetrahydrothienyl and piperidinyl.
The compounds of formula (I) are useful inhibitors of enzymes involved in the degradation of fats. Preferably therefore the first aspect of the invention provides the use of a compound of formula (I) as defined hereinabove, or a pharmaceutically acceptable salt, ester, amide or prodrug thereof, in the manufacture of a medicament for the control or treatment of obesity, or obesity-related disorders or for promoting non-medical weight loss.
Preferably, a compound for use according to the first aspect of the invention is a compound of formula (II): 
or a pharmaceutically acceptable salt, ester, amide or prodrug therof, wherein:xe2x80x94
R1, R4, R5, R6, R7, X1 and X2 are as defined above for formula (I); and
R8, R9, R10, R11 are each independently hydrogen, halo, hydroxy, amino, nitro, cyano,
or a group R1, as defined above,
or a group R12Q where Q is O, CO, CONH, NHCO, S, SO, SO2, or SO2NH2 and R12 is hydrogen or a group R1 as defined above,
or a group R1R2N where R1 is as defined above and R2 is hydrogen or R1, with the proviso that any hetero atom substituent in R1 and/or R2 must be separated from the aromatic hetero atom substituent by at least two carbon atoms (preferably saturated). Preferred values of R1, R8, R9, R10 and R11 are as defined below for compounds of formula (IIa).
More preferably, a compound for use according to the first aspect of the invention comprises a compound of formula (II), or a pharmaceutically acceptable salt, ester, amide or prodrug therof; wherein:
R1 is either a branched or unbranched alkyl group having up to 25, e.g. up to 20 carbon atoms, an aryl (e.g. optionally substituted phenyl or 2-naphthyl), an arylalkyl group wherein the alkyl moiety has up to 25, e.g. up to 20 carbon atoms, or an aryl aryl group, wherein the aryl alkyl group or the aryl aryl group may be separated by a spacer, and where the spacer can be one or more of an ester, amide, O, CH2 or a ketone and wherein any aryl group is preferably a phenyl, optionally substituted with alkyl, haloalkyl or halogen;
R8 is hydrogen or fluorine;
R9 is lower branched or unbranched alkyl having 1 to 10 carbon atoms, preferably methyl; cyclic alkyl having 3 to 10 carbon atoms, preferably cyclopropyl; haloalkyl, preferably trifluoromethyl; or a halogen, most preferably chlorine or fluorine;
R10 is hydrogen lower branched or unbranched alkyl having 1 to 10 carbon atoms, preferably methyl; cyclic alkyl having 3 to 10 carbon atoms, preferably cyclopropyl; haloalkyl, preferably trifluoromethyl; or a halogen, most preferably chlorine or fluorine;
R11 is hydrogen lower branched or unbranched alkyl having 1 to 10 carbon atoms, preferably methyl, or halogen, preferably fluorine.
In particular, R1 is an unbranched alkyl group, having 12, 14, 15, 16, 17 or 18 carbon atoms in the alkyl chain. In addition to this particular option for R1, R9 may be methyl.
In a second aspect the present invention provides novel compounds of formula (IIa): 
or a pharmaceutically acceptable salt, ester, amide or prodrug thereof; where:
R1a is
(i) a C10-30 branched or unbranched alkyl, optionally substituted by one or more independently of C3-6 cycloalkyl, C3-6 cycloalkenyl, aryl, heteroaryl, reduced heteroaryl, xe2x80x94C(O)R13, xe2x80x94CO2R13, xe2x80x94SOR13, SO2R13, xe2x80x94NR13R14, xe2x80x94OR13, xe2x80x94SR13, xe2x80x94C(O)NR13R14, xe2x80x94NR14C(O)R13, halogen, cyano, and nitro and/or optionally interrupted by one or more oxygen atoms with the proviso that any hetero atom in R1 must be separated from the exocyclic oxygen atom (or from any other heteroatom) by at least two carbon atoms (preferably saturated);
(ii) C2-25 alkenyl, C2-25 alkynyl, C3-6 cycloalkenyl, aryl-C2-25 alkenyl, heteroaryl-C2-25alkenyl, reduced heteroaryl, reduced heteroaryl-C1-25 alkyl or a substituted derivative of any of the foregoing groups wherein the substituents are one or more independently of C1-6 alkyl, halosubstituted C1-6 alkyl, aryl, aryl-C1-6 alkyl, heteroaryl, reduced heteroaryl, reduced heteroaryl-C1-6 alkyl, C1-6 alkoxy, aryl-C1-6 alkoxy, xe2x80x94C(O)R13, xe2x80x94CO2R13,xe2x80x94SOR13, xe2x80x94SO2R13, xe2x80x94NR13R14, xe2x80x94OR13, xe2x80x94SR13, xe2x80x94C(O)NR13R14, xe2x80x94NR14C(O)R13, halogen, cyano, and nitro, with the proviso that any hetero atom in R1 must be separated from the exocyclic oxygen atom (or from any other heteroatom) by at least two carbon atoms (preferably saturated);
(iii) a C1-9 alkyl group interrupted by one or more oxygen atoms and optionally substituted by one or more independently of C3-6 cycloalkyl, C3-6 cycloalkenyl, aryl, heteroaryl, reduced heteroaryl, xe2x80x94C(O)R13, xe2x80x94CO2R13, xe2x80x94SOR13, xe2x80x94SO2R13, NR13R14, OR13, SR13, xe2x80x94C(O)NR13R14, xe2x80x94NR14C(O)R13, halogen, cyano and nitro with the proviso that any hetero atom in R1 must be separated from the exocyclic oxygen atom (or from any other heteroatom) by at least two carbon atoms (preferably saturated); or
(iv) a C1-9 alkyl group substituted by a group selected from xe2x80x94C(O)R13, xe2x80x94CO2R13, SOR13, SO2R13, NR13R14, OR13, SR13, C(O)NR13R14, NR14C(O)R13; halogen, cyano, nitro, bicyclic aryl, bicyclic heteroaryl, monocyclic or bicyclic reduced heteroaryl, and monocyclic heteroaryl other than imidazolyl;
(v) a phenyl group substituted by a group selected from OR17, xe2x80x94COR13, xe2x80x94CO2R13, SOR13, SO2R13, CONR13R14, NR14C(O)R13; halosubstituted C1-6 alkyl, aryl, arylC1-6alkyl, heteroaryl and heteroarylC1-6alkyl; or
(vi) a bicyclic aryl, bicyclic heteroaryl, monocyclic or bicyclic reduced heteroaryl, or monocyclic heteroaryl group other than imidazolyl, optionally substituted by a group selected from OR17, xe2x80x94COR13, xe2x80x94CO2R13, SOR13, SO2R13, CONR13R14, NR14C(O)R13; halosubstituted C1-6 alkyl, aryl, arylC1-6alkyl, heteroaryl and heteroarylC1-6alkyl;
where R13 and R14 each independently represents hydrogen, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-6cycloalkyl, C3-6cycloalkenyl, aryl, arylC1-10alkyl, heteroaryl, heteroarylC1-10alkyl, reduced heteroaryl or reduced heteroaryl, C1-10alkyl, and R17 represents hydrogen or C2-10alkenyl, C2-10alkynyl, C3-6cycloalkyl, C3-6cycloalkenyl, aryl, arylC1-10alkyl, heteroaryl, heteroarylC1-10alkyl, reduced heteroaryl or reduced heteroaryl, C1-10alkyl
and R8a, R9a, R10a and R11a are each independently hydrogen, halo, hydroxy, amino, nitro, cyano, thiol, C1-10alkyl, C1-10alkoxy, C1-10cycloalkyl, C1-10cycloalkoxy, C(O)R15, C(O)NR15R16, S(O)R15 or haloC1-10alkyl;
where R15 and R16 each independently represent hydrogen or C1-10alkyl.
In compounds of formula (IIa):
When R1a represents a group defined in (i) this is preferably a C10-25 e.g. a C10-20 branched or unbranched alkyl group, advantageously a C12-18 e.g. a C14-18 branched or unbranched alkyl group, optionally interrupted by one or more oxygen atoms. Optional substituents for said alkyl groups are preferably selected from one or more of aryl e.g. phenyl; heteroaryl e.g. thienyl; aryloxy, e.g. phenoxy; heteroaryloxy, e.g. benzoxazinyloxy; xe2x80x94CO2R13 e.g. COOH; NR13R14 e.g. NH2; xe2x80x94CONR13R14 e.g. CONHCH3; cyano, nitro, halogen and OH. R13 and R14 preferably each independently represent hydrogen or C1-6alkyl.
When R1a represents a group defined by (ii) this is preferably a C10-25 e.g. a C10-20 branched or unbranched alkenyl or alkynyl group, advantageously a C14-18 branched or unbranched alkenyl or alkynyl group. Preferred optional substituents are those defined as preferred substituents in group (i).
When R1a represents a group defined by (iii) the C1-9 group preferably contains one or two oxygen atoms. Preferred optional substituents are as defined above for groups (i) and (ii).
When R represents a group defined by (iv) preferred substituents are as defined above for groups (i)-(iii).
When R1a represents a group defined by (v) preferred substituents are selected from OR17, CO2R13, C(O)NR13R14, NR14C(O)R13 and aryl C1-10alkyl. The phenyl group R1a and any additional phenyl group or moiety in the substituent may also be substituted by one or more of halogen, alkyl or haloalkyl.
When R1a represents a group defined by (vi) this is preferably selected from naphthyl, pyridyl, pyrrolyl and piperidinyl.
R1a is preferably selected from groups (i) and (v) defined above.
R1a is preferably C10-20 branched or unbranched alkyl, optionally interrupted by one or two oxygen atoms
and/or optionally substituted by one or more of aryl, eg. phenyl; aryloxy e.g. phenoxy wherein the phenyl moiety may be optionally substitued by alkyl, haloalkyl, halogen or phenoxy; heteroaryl, eg. thienyl; heteroaryloxy e.g. benzoxazinyloxy (which may optionally carry an oxo substituent); cyano, nitro, xe2x80x94CO2R13, xe2x80x94NR13R14, xe2x80x94CONR13R14, OH and halogen.
R1a is also advantageously phenyl substituted by one or more, but most preferably one, of OR17, xe2x80x94CO2R13, xe2x80x94C(O)NR13R14, NR14C(O)R13 and aryl C1-10alkyl.
R13 and R14 preferably each independently represent hydrogen or C1-6alkyl.
R17 preferably represents phenyl, optionally substituted by alkyl, haloalkyl, halogen or phenoxy, wherein the phenyl moiety may also be optionally substituted by alkyl, haloalkyl or halogen.
Most preferably R1a is an unbranched alkyl chain having 14, 15, 16, 17 or 18 carbon atoms.
R8a is preferably hydrogen or halogen eg. fluorine; most preferably hydrogen.
R9a is preferably hydrogen or lower branched or unbranched alkyl having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, preferably methyl, cyclic alkyl having 3 to 6 carbon atoms, preferably cyclopropyl, haloC1-6alkyl, preferably trifluoromethyl or a halogen, preferably chlorine or fluorine;
R10a is preferably hydrogen, lower branched or unbranched alkyl having 1 to 10 carbon atoms, preferably 1-6 carbon atoms preferably methyl, cyclic alkyl having 3 to 6 carbon atoms preferably cyclopropyl, haloC1-6alkyl preferably trifluoromethyl or a halogen preferably chlorine or fluorine;
R11a is preferably hydrogen, halogen, eg. fluorine; or branched or unbranched alkyl having 1 to 10 carbon atoms, preferably 1-6 carbon atoms, eg. methyl.
Preferably, in compounds of formula (IIa) at least one of R8a, R9a, R10a and R11a represents a substituent other than hydrogen. Thus, for example, R8a may represent a hydrogen atom and R9a, R10a and R11a are as defined above. In a further preferred embodiment each of R8a and R11a represents a hydrogen atom, R9a represents a substituent as defined above and R10a represents a hydrogen atom or a substituent. In a further embodiment R8a, R9a and R10a represent hydrogen and R11a represents a substituent as defined above, eg. methyl. Most preferably each of R8a, R10a and R11a represents a hydrogen atom, and R9a represents a substituent as defined above, advantageously a C1-6alkyl group, especially methyl.
A further group of novel compounds within the scope of formula (II) is that wherein R1 is as defined for formula (II) and at least one of R8, R9, R10 and R11 represents a C8-20 alkyl group, preferably a C8-10 alkyl group. Most preferably in this embodiment either R9 or R10 represents a C8-10 alkyl group and the remaining substituents on the benzene ring are all hydrogen.
Examples of pharmaceutically acceptable salts of the above compounds include those derived from organic acids such as methanesulphonic acid, benzenesulphonic acid and p-toluenesulphonic acid, mineral acids such as hydrochloric and sulphuric acid and the like, giving methanesulphonate, benzenesulphonate, p-toluenesulphonate, hydrochloride and sulphate, and the like, respectively or those derived from bases such as organic and inorganic bases. Examples of suitable inorganic bases for the formation of salts of compounds for this invention include the hydroxides, carbonates, and bicarbonates of ammonia, lithium, sodium, calcium, potassium, aluminium, iron, magnesium, zinc and the like. Salts can also be formed with suitable organic bases. Such bases suitable for the formation of pharmaceutically acceptable base addition salts with compounds of the present invention include organic bases which are nontoxic and strong enough to form salts. Such organic bases are already well known in the art and may include amino acids such as arginine and lysine, mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine, choline, mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and trimethylamine, guanidine; N-methylglucosamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; tris(hydroxymethyl) aminomethane; and the like.
Salts may be prepared in a conventional manner using methods well known in the art. Acid addition salts of said basic compounds may be prepared by dissolving the free base compounds according to the first or second aspects of the invention in aqueous or aqueous alcohol solution or other suitable solvents containing the required acid. Where a compound of formula (I) contains an acidic function a base salt of said compound may be prepared by reacting said compound with a suitable base. The acid or base salt may separate directly or can be obtained by concentrating the solution eg. by evaporation. The compounds of this invention may also exist in solvated or hydrated forms.
The invention also extends to prodrugs of the aforementioned compounds. A prodrug is commonly described as an inactive or protected derivative of an active ingredient or a drug which is converted to the active ingredient or drug in the body.
Representative compounds according to the first and/or second aspects of the invention are those which include:
Apart from compounds 1-3, 6, 7 and 9, the compounds of Table 1 are believed to be novel and as such form preferred embodiments of the present invention.
Preferred compounds of formula (II) listed in Table 1 include compounds numbers 4, 5, 8, 10, 11, 12, 13, 14, 21, 26, 27, 28, 30, 32, 33, 34, 35 and 78.
These specific compounds are believed to be novel and as such form a further aspect of the invention.
Preferred compounds of formula (IIa) listed in Table 1 include compounds numbers 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 29, 31, 36, 37, 38, 39, 79, 80, 81, 82, 84 and 85.
Particularly preferred compounds of formula (IIa) are:
15: 2-Decyloxy-6-methyl-4H-3,1-benzoxazin-4-one
16: 6-Methyl-2-tetradecyloxy-4H-3,1-benzoxazin-4-one
18: 2-Hexadecyloxy-6-methyl-4H-3,1-benzoxazin-4-one
Preferred compounds of the invention listed above extend to the tautomers thereof, as well as (but not limited to) pharmaceutically acceptable salts, esters, amides or prodrugs thereof or a derivative optionally with one or more lipid groups (natural or synthetic) attached.
A third aspect of the invention provides a process for the manufacture of any one or more of the novel compounds or derivatives according to the first or second aspects of the invention. Thus, the present invention provides a process for the preparation of a novel compound of formula (II) in particular a compound of formula (IIa) which process comprises:
Process (A) Reacting a Compound of Formula (IV): 
with a compound of formula (V): 
or
Process (B) Cyclising a Compound of Formula (VI) 
wherein R1 and R8-R11 are as hereinbefore defined and R18 is hydrogen or C1-6alkyl.
or:
Process (C) Reacting a Compound of Formula (VII) 
with a compound of formula (VIII):
R1OHxe2x80x83xe2x80x83(VIII)
or:
Process (D) Converting a Compound of Formula (I), (II) or (IIa) into a Different Compound of Formula (IIa), by, for Example,
(i) reduction of a compound of formula (IIa) wherein any of R1, R8, R9, R10 and R11 contains an alkenyl or alkynyl group or moiety, to the corresponding alkyl or alkenyl group or moiety; or
(ii) alkylation of a compound of formula (IIa) where one or more of R8, R9, R10 and R11 represents a halogen atom.
Process (A) may be effected by reacting a compound of formula (IV) with a chloroformate of formula (V). The process is preferably carried out under basic conditions, e.g. using pyridine. The reaction may be cooled to avoid overheating. The resulting carbamate intermediate is then cyclised by reaction with excess chloroformate or by addition of another cyclisation reagent, which promotes ring closure. Suitable cyclisation reagents include for example, methyl chloroformate, carbonyl diimidazole, acetic anhydride, phosgene, oxalyl chloride, thionyl chloride or a peptide coupling agent such as dicyclohexyl carbodiimide (DCC). The cyclisation reagent is preferably phosgene, triphosgene or thionyl chloride. It will be understood by those skilled in the art that when a chloroformate is used to effect the cyclisation, this may be provided by employing an excess of the compound of formula (V). Preferably, however, a low molecular weight chloroformate is employed, on grounds of cost and ease of removing the resulting alcohol.
Compounds of formula (V) for use in the process (A) may be prepared by standard methods well known in the art, e.g. by reaction of the corresponding alcohol R1OH with phosgene in a solvent such as toluene. The product may be isolated in conventional manner by removal of solvent and volatile by-products.
Process (B) may be effected by reaction of a compound (VI) wherein R18 is hydrogen, in the presence of a cyclisation reagent, e.g. an alkyl chloroformate, for example as described for process (A). Alternatively a compound (VI) may be cyclised by treatment with a dehydrating agent such as concentrated sulphuric acid.
Compounds (VI) wherein R18 is an alkyl group may be prepared by reacting an ester corresponding to formula (IV) with e.g. phosgene and a base such as pyridine to afford the corresponding isocyanate, followed by treatment with an alcohol R1OH. If desired the ester (i.e. where R18 is alkyl) may be hydrolysed to the corresponding acid (R18=H) using for example lithium hydroxide in e.g. aqueous tetrahydrofuran or aqueous dioxane.
It will be appreciated that process (A) also proceeds via an intermediate of formula (VI) and is hence a variant of process (B).
Process (C) may be effected by reacting a compound of formula (VII) with an alcohol R1OH in the presence of a base, e.g. Hunig""s base (diisopropylethylamine). A compound of formula (VII) may be prepared from the corresponding anhydride of formula (IX) 
by reaction with for example phosphorus oxychloride (POCl3) at elevated temperature e.g. 100xc2x0 C.
The anhydride of formula (IX) may itself be obtained by cyclisation of a compound of formula (IV), for example using phosgene or a synthetic equivalent.
In process (D), reduction of an alkenyl or alkynyl group may be effected for example by catalytic hydrogenation using e.g. 10% palladium on charcoal in an alcoholic solvent, such as ethanol, under 1 atmosphere of hydrogen gas.
Alkylation according to process (D)(ii) may be effected using a Stille or other palladium catalysed cross-coupling process, using e.g. tetra-alkyl tin such as tetramethyl tin and PhCH2Pd(PPh3)2Cl in HMPA at elevated temperature e.g. 50-100xc2x0 C. Other halides or pseudohalides e.g. triflates may be employed as starting materials.
A fourth aspect of the invention is a compound according to the first and second aspects of the invention (i.e. compounds of formulae (I), (II) and (IIa)), for use in medicine. Preferred features of the first and second aspects of the invention also apply to the fourth aspect. Further details of the fourth aspect of the invention are set out in the text which follows.
A fifth aspect of the invention relates to a compound according to the first and/or second aspects of the invention for use in the inhibition of an enzyme whose preferred mode of action is to catalyse the hydrolysis of an ester functionality. This includes both in vivo and in vitro uses and other uses such as industrial uses. Such an enzyme is one which catalyses the breakdown of a substrate containing an ester functionality by the addition of water, resulting in the cleavage of a chemical bond. Such enzymes are involved in key processes in the body. Enzymes according to this invention include lipases (hydrolyse fatty acid esters), esterases (hydrolyse esters) and phosphatases (hydrolyse phosphate esters).
The enzyme is preferably a lipase. Lipases include pancreatic lipase, gastric lipase, lipoprotein lipase, lingual lipase, adipose tissue lipase, hormone sensitive lipase, phospholipase A1, A2, B, C, D etc., hepatic lipase, and other triacyl, diacyl and monoacylglycerol lipases in the mammalian body. Many similar such lipases are also known in plants, fungi and microorganisms.
Also covered are esterase enzymes and phosphatase enzymes. Esterase enzymes include pig liver esterase, cholesteryl esterase, retinyl esterase, 1-alkyl-2-acetylglycerophosphocholine esterase, carboxylic ester hydrolases, and cholesterol esterase. Phosphatase enzymes include serine/threonine phosphatases PP1, PP2 and PP3, phosphoprotein phosphatase, myosin-light-chain phosphatase, protein phosphoprotein 2C, and protein tyrosine phosphatase.
Compounds according to the invention, for use in medicine, are primarily for use in relation to the prevention and/or treatment of a medical condition such as obesity, hyperlipaemia, hyperlipidaemia and related diseases such as hyperglycaemia (type II diabetes), hypertension, cardiovascular disease, stroke, gastrointestinal disease and gastrointestinal conditions. Compounds according to the first and second aspect of the invention are useful in these and other conditions due to their ability to inhibit an enzyme whose preferred mode of action is to catalyse the hydrolysis of an ester functionality (in vivo, as the enzyme naturally occurs). The invention also relates to non-medical weight loss, such as cosmetic weight loss and includes improving bodily appearance in general. Throughout this text, the prevention and/or treatment of any disorder means any effect which mitigates any damage or any medical disorder, to any extent, and includes prevention and treatment themselves. The term xe2x80x9ctreatmentxe2x80x9d means any amelioration of disorder, disease, syndrome, condition, pain or a combination of two or more thereof.
Clearly, an important application of the invention is in relation to weight loss (of all kinds as described above) in humans. However, the invention applies to medical and non-medical weight loss in any animal whose metabolism of fat and fat derivatives involves an enzyme whose preferred mode of action is to catalyse the hydrolysis of an ester functionality (in vivo, as the enzyme naturally occurs). Thus, the invention has veterinary application and is particularly useful in relation to medical and non-medical weight loss in companion animals such as pet cats and dogs as well as in animals which provide meat for human consumption. In the case of the latter, the application of the present invention is to reduce fat content in order to provide a leaner meat product.
It is also believed that the compounds may be useful in reducing levels of toxins (e.g. dioxins and PCBs) stored in body fat. Without wishing to be bound by theory, it is believed that increasing the amount of undigested fat passing through the body enhances diffusion of toxins from fat stored in the body into fats in the blood, and thence into the intestine.
The fifth aspect of the invention has important applications. It includes test and diagnostic methods and the control and inhibition of unwanted enzymes, preferably lipases, in any process or in any product. The processes or products, which preferably involve a lipase, include: processing of agricultural commodities (e.g. oilseeds), recovery and isolation of enzymes from biotechnological processes (e.g. involving lysis of microorganisms), the manufacture and extraction of crude oil (especially oil and plastics), the industrial manufacture of triglycerides or other fats, manufacture of healthcare goods which comprise surfactants, soap or detergent (e.g. bath oils, creams), the manufacturing and processing of liposomes (e.g. healthcare products, diagnostics, gene therapy), the treatment of industrial waste (e.g. paper effluent treatment) and preventing the degradation of foodstuff which comprises a fat (e.g. chocolate processing). Thus, the invention also relates to these products and processes, e.g. a foodstuff which comprises a compound according to the first aspect of the invention, in particular foodstuffs which have a high fat content such as cakes, biscuits, pastry-products and the like and chocolate products. The preferred features of the fifth aspect of the invention, including an enzyme whose preferred mode of action is to catalyse the hydrolysis of an ester functionality (in vivo, as the enzyme naturally occurs) are as discussed for the previous aspects of the invention.
A sixth aspect of the invention provides a composition comprising a novel compound according to the first or second aspect of the invention, in combination with a pharmaceutically acceptable carrier or diluent. Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile). The composition may be a mixed preparation of a composition or may be a combined preparation for simultaneous, separate or sequential use (including administration).
The compounds according to the invention for use in the aforementioned indications may be administered by any convenient method, for example by oral (including by inhalation), parenteral, mucosal (e.g. buccal, sublingual, nasal), rectal or transdermal administration and the compositions adapted accordingly.
For oral administration, the compounds can be formulated as liquids or solids, for example solutions, syrups, suspensions or emulsions, tablets, capsules and lozenges.
A liquid formulation will generally consist of a suspension or solution of the compound or physiologically acceptable salt in a suitable aqueous or non-aqueous liquid carrier(s) for example water, ethanol, glycerine, polyethylene glycol or an oil. The formulation may also contain a suspending agent, preservative, flavouring or colouring agent.
A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and microcrystalline cellulose.
A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, powders, granules or pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
Compositions for oral administration may be designed to protect the active ingredient against degradation as it passes through the alimentary tract, for example by an outer coating of the formulation on a tablet or capsule.
Typical parenteral compositions consist of a solution or suspension of the compound or physiologically acceptable salt in a sterile aqueous or non-aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.
Compositions for nasal or oral administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal once the contents of the container have been exhausted. Where the dosage form comprises an aerosol dispenser, it will contain a pharmaceutically acceptable propellant. The aerosol dosage forms can also take the form of a pump-atomiser.
Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.
Compositions for rectal or vaginal administration are conveniently in the form of suppositories (containing a conventional suppository base such as cocoa butter), pessaries, vaginal tabs, foams or enemas.
Compositions suitable for transdermal administration include ointments, gels, patches and injections including powder injections.
Conveniently the composition is in unit dose form such as a tablet, capsule or ampoule.
The compositions of the sixth aspect of the invention are useful in the prevention and/or treatment of obesity, obesity-related disorder, other medical weight loss and non-medical related weight loss. Preferred features of this aspect of the invention are as described above for the first to fifth aspects of the invention.
A seventh aspect of the invention provides a process for the manufacture of a composition according to the sixth aspect of the invention. The manufacture can be carried out by standard techniques well known in the art and involves combining a compound according to the first or second aspect of the invention and the pharmaceutically acceptable carrier or diluent. The composition may be in any form including a tablet, a liquid, a capsule, and a powder or in the form of a food product, e.g. a functional food. In the latter case the food product itself may act as the pharmaceutically acceptable carrier.
An eighth aspect of the invention provides a method for the prevention and/or treatment of obesity or an obesity-related disorder, the method comprising the administration of a compound according to the first or second aspect of the invention, preferably in combination with a pharmaceutically acceptable carrier or diluent (as per the sixth aspect of the invention). Obesity-related disorders include hyperlipeamia, hyperlipideamia, hyperglycaemia, hypertension, cardiovascular disease, stroke, gastrointestinal disease and gastrointestinal conditions. The compound or composition is preferably administered to a patient in need thereof and in a quantity sufficient to prevent and/or treat the symptoms of the condition, disorder or disease For all aspects of the invention, particularly medical ones, the administration of a compound or composition has a dosage regime which will ultimately be determined by the attending physician and will take into consideration such factors such as the compound being used, animal type, age, weight, severity of symptoms, method of administration, adverse reactions and/or other contraindications. Specific defined dosage ranges can be determined by standard design clinical trials with patient progress and recovery being fully monitored. Such trials may use an escalating dose design using a low percentage of the maximum tolerated dose in animals as the starting dose in man.
The physiologically acceptable compounds of the invention will normally be administered in a daily dosage regimen (for an adult patient) of, for example, an oral dose of between 1 mg and 2000 mg, preferably between 30 mg and 1000 mg, e.g. between 10 and 250 mg or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 50 mg, e.g. between 1 and 25 mg of the compound of the formula (I) or a physiologically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day. Suitably the compounds will be administered for a period of continuous therapy, for example for a week or more.
A ninth aspect of the invention provides a cosmetic method (non-therapeutic) for maintaining a given weight, or for cosmetic weight loss, the method comprising the administration of a compound according to the first or second aspect of the invention, preferably in combination with a pharmaceutically acceptable carrier or diluent (as per the sixth aspect of the invention). The compound or composition is preferably administered to a subject in need or in desideratum thereof and in a quantity sufficient to maintain a given weight or for cosmetic weight loss.
The eighth and ninth aspects of the invention relate to methods which are applicable to humans and other animals, in particular companion animals (such as dogs and cats) and other animals which provide meat for human consumption, such as cattle, pigs and sheep (all of any age).
The invention will now be described with reference to the following non-limiting examples.
Biological Test Methods and Results
Test Compounds
The benzoxazinone compounds used in the following tests are identified by the reference number assigned in Table 1 hereinbefore.
Measurement of Lipase Activity Using a Quinine Diimine Dye Calorimetric Assay
The inhibitory activity of the selected compounds to pancreatic lipase was measured in the following assay available from Sigma Ltd (Lipase-PS(trademark), catalog number 805-A): 
The glycerol released from the action of pancreatic and monoglyceride lipase was oxidised to release H2O2. The peroxidase reaction step then produces a quinine dye which is pink in colour and absorbs light at a wavelength of 550 nm.
Inhibitor
Individual test compounds were dissolved in DMSO (dimethyl sulphoxide) at 10 mM. DMSO was used to avoid any problems with compounds being water-insoluble. For individual compounds, the IC50 (concentration at which lipase activity is inhibited to one half of the maximum) was calculated by measuring the inhibitory activity from log-dose response curves using a range of inhibitor concentrations.
Results
A range of compounds was assayed in the quinine diimine dye calorimetric assay which provides a rapid method to measure lipase inhibitory activity. None of the compounds tested interfered with the calorimetric reaction, i.e. they did not give false positive results.
A range of inhibitory activities for the tested benzoxazinone compounds was observed, indicating that these compounds are inhibitors of human pancreatic lipase. The following compounds had an IC50 of xe2x89xa6100 nM: Compounds 9, 11-16, 18-22, 23, 24-39, 77, 78, 79-82, 84 and 85.
Measurement of Lipase Enzyme Activity Using a NaOH Titration Method
The inhibitory activity of the selected compounds to pancreatic lipase was measured in the assay described in Pasquier et al; 1996, Vol 7, Nutritional Biochemistry, 293-302.
Log dose/response curves were constructed using a range of inhibitor concentrations.
Results
Selected benzoxazinone compounds were tested in the NaOH titration assay. In this assay, the activity of porcine pancreatic lipase in a system containing lipid micelles is recorded. These conditions are therefore similar to those encountered in the gastrointestinal tract.
A range of inhibitory activities were observed for the tested benzoxazinone compounds in this assay, indicating that these compounds are inhibitors of porcine pancreatic lipase. The following compounds had IC50""s of xe2x89xa61 xcexcM:
Compounds 1, 2, 4, 6-9, 11-16, 18, 22-25, 27-36, 37-39, 78, 82, 84 and 85.
The results demonstrate that a number of selected benzoxazinones are inhibitors of fat digestion and that these compounds may be particularly suitable for the treatment of obesity.
Measurement of Trypsin Activity
Porcine trypsin (Boehringer) was dissolved at a concentration of 1 mg/ml in 100 mM MOPS (3-[N-Morpholino]propanesulphonic acid) pH 7.3 containing 2 mM CaCl2. Prior to use, the enzyme was diluted 500 times to give a final concentration of 2 xcexcg/ml.
Selected compounds were routinely stored as 5 mM stock solutions dissolved in DMSO (Dimethylsulphoxide) at xe2x88x9220xc2x0 C. For the assay, aliquots were defrosted and a series of dilutions (xc3x97100, xc3x97200, xc3x971,000, xc3x972,000, xc3x9710,000, xc3x9720,000 and xc3x97100,000) made in 100 mM MOPS pH 7.3 containing 2 mM CaCl2. The substrate Bz-Phe-Val-Arg-pNA (Benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide) was dissolved in DMSO to give a 10 mM solution. Immediately prior to use, the substrate was diluted to 0.3 mM (30 xcexcl/ml) in 100 mM MOPS containing 2 M CaCl2.
The assay was set up in triplicate in a 96 well ELISA plate. 10 xcexcl 2 xcexcg/ml trypsin, 26 xcexcl diluted inhibitor and 190 xcexcl substrate were added sequentially. The plates were then incubated at 37xc2x0 C. in a BioRad Benchmark Microplate Reader. The rate of release of p-nitroaniline was measured at 405 nM over 10 minutes relative to that of the enzyme without inhibitor.
Measurement of Chymotrypsin Activity
Bovine Chymotrypsin (Sigma Type II Cat. No. C4129) was dissolved at a concentration of 1 mg/ml in 100 mM Tris pH 7.8. Prior to use, the enzyme was diluted 20 fold immediately before use, with the same buffer.
Selected compounds were routinely stored as 5 mM stock solutions dissolved in DMSO (Dimethylsulphoxide) at xe2x88x9220xc2x0 C. For the assay, aliquots were defrosted and a series of dilutions (xc3x9720, xc3x97100, xc3x97200, xc3x971,000, xc3x972,000, xc3x9710,000, xc3x9720,000 and xc3x97100,000) made in 100 mM Tris pH 7.8. The substrate H-Ala-Ala-Phe-p-nitroanilide (H-alanyl-alanyl-phenylalanine-p-nitroanilide) (Bachem Cat. No. L-1095) was dissolved in DMSO to give a 10 mM stock and stored at 4xc2x0 C. until use. Immediately prior to use, the substrate was diluted to 0.3 mM final concentration (30 xcexcl/ml) just before use.
The assay was set up in triplicate in a 96 well ELISA plate. 10 xcexcl 50 xcexcg/ml chymotrypsin, 50 xcexcl diluted inhibitor and 190 xcexcl substrate were added sequentially. The plates were then incubated at 37xc2x0 C. in a BioRad Benchmark Microplate Reader. The rate of release of p-nitroaniline was measured at 405 nM over 10 minutes relative to that of the enzyme without inhibitor.
Results
The selectivity of compound 18 towards human pancreatic lipase, trypsin and chymotrypsin was measured in the assays described above. The inhibitory activity of compound 18 towards lipase was assessed in the quinine diimine dye calorimetric assay.
At 500 nM compound 18 had inhibited 98.7% of the pancreatic lipase activity but only 4% of the trypsin activity and 12.5% of the chymotrypsin activity. Thus compound 18 is a highly selective lipase inhibitor.
Mouse Model Assay
Compound 18 was assayed in a mouse model as described by Isler et al., British Journal of Nutrition, 1995, 73:851-862 and was found to be a potent lipase inhibitor.
Synthesis of Intermediates
Synthesis of 4-substituted Anthranilic Acids